Journal Mejia

Phenolic Content and Antioxidant Activity of Selected Philippine Traditional Medicinal Plants Used to Treat Gastro-intestinal Ailments Karissa Grace F. Mejiaa, Flordeliza C. de Verab a

School of Chemical Engineering and Chemistry, Mapua Institute of Technology, Manila, Republic of Philippines School of Chemical Engineering and Chemistry, Mapua Institute of Technology, Manila, Republic of Philippines

b

Abstract Medicinal plants have been an important part of indigenous medical systems in the Philippines and frequent consumption of these plants is associated with preventing and curing different types of diseases. This has been attributed to the presence of various forms of antioxidants like polyphenol compounds including flavonoids. Six plants of the traditional medicine for gastrointestinal disorders in the Philippines specifically Tsaang gubat, Banaba, Malunggay, Tubang-bakod, Sambong and Mangostan were analyzed for their antioxidant activity, total polyphenol content (TPP), total flavonoid content (TFC) and antimicrobial assay. The TPP of the extracts was determined spectrophotometrically by the Folin-Ciocalteau method. The TFC was obtained using the AlCl3 method whereas the antioxidant activity was measured using the 2-2-diphenyl-1-picrylhydrazyl (DPPH) assay. The antimicrobial assay was determined by the agar cup method using Escherichia coli and Staphylococcus aureus as the test organisms which are common causes of diarrhea. Methanolic extracts of tsaang gubat exhibited the highest TPP, TFC, and DPPH Radical Scavenging Activity (RSA). Sambong showed the lowest TPP and DPPH RSA, whereas tubang-bakod showed the lowest TFC. Most of the methanolic extracts showed antimicrobial activity against Escherichia coli and had slightly inhibitory effects on Staphylococcus aureus. Keywords: antimicrobial activity, antioxidant activity, flavonoid, gastro-intestinal ailments, polyphenols

I.

Introduction

Plants have formed the basis of sophisticated traditional medicine systems that have been in existence for thousands of years and which continue to provide mankind with new remedies. Although some of the therapeutic properties attributed to plants have proven to be erroneous, medicinal plant therapy is based on the empirical findings of hundreds and thousands of years (Gurub-Fakim, 2006). The interest in the study of medicinal plants as a source of pharmacologically active compounds has increased worldwide. It is recognized that in some developing countries, where living conditions are crowded and hygiene is poor, plants are the main medicinal source to treat infectious diseases and where diarrhea and dysentery caused by bacterial enteropathogens are among the main causes of morbidity and mortality (Alanis et al., 2005). In the Philippines, there is an astounding variety of medicinal plants that have been reported for their folkloric medicinal purposes but only a few medicinal plants have been studied and documented for their functions and uses. In recent years, interest has been shifted to antioxidants from various plants. *Corresponding author Email address: [email protected]

Antioxidants are substances that can prevent or delay the oxidative damage of lipids, proteins and nucleic acids by reactive oxygen species, which include reactive free radicals such as superoxide, hydroxyl, peroxyl, alkoxyl and non-radicals such as hydrogen peroxide, hypochlorous, etc. They scavenge radicals by inhibiting initiation and breaking chain propagation or suppressing the formation of free radicals by binding to the metal ions, reducing hydrogen peroxide, and quenching superoxide and singlet oxygen (Lim et. al, 2007). Antioxidant sources like medicinal plants are used as herbal tea drinks, as body applications and for washing sores and wounds, are medicinal for asthma, dysentery, skin diseases, cancer, wounds, boils, stomach troubles, colds and other pulmonary diseases. There are fruits that help prevent such illnesses as diabetes, high blood pressure, respiratory ailments and blood hemorrhage. There are leaves used as medicinal application for wounds, broken bones and others. There are culinary herbs and spices that also help prevent disease like gastrointestinal problems. Studies on the antioxidant capacity and phenolic content of Philippine medicinal plants are limited. There is a need for further studies on the properties of the medicinal plants to validate their use in traditional medicine. There are a lot of studies related to the common medicinal plants in other countries but a different kind of environment would greatly affect the

growth of these plants as well as their benefits to humans. The quantity and quality of phenolic phytochemicals present in fruits and vegetables are significantly influenced by cultivar, environment, soil type, and growing and storage conditions (Singh et al., 2009). The objective of the study was to determine the total antioxidant activity, phenolic content, flavanoid content and antimicrobial activity of selected common Philippine medicinal plants that are intended for gastrointestinal disorders specifically diarrhea, such as the leaves of queen's crape myrtle (Lagerstroemia speciosa, banaba), drumstick (Moringa oleifera, malunggay), physic nut (Jatropha curcas, tubang-bakod), blumea camphor (Blumea balsamifera, sambong) and mangosteen (Garcinia mangostana, mangostan). The results of this study give scientific support to the use of selected medicinal plants in the Philippines for the treatment of gastrointestinal disorders such as diarrhea and establish renewable sources of antioxidants from plants. The analysis of the polyphenol content, flavonoid content, antioxidant activity and antimicrobial activity used only the leaves of the medicinal plant. The DPPH radical scavenging method was used in determining the total antioxidant activity, the Folin-Ciocalteu method for the total polyphenol content and the AlCl3 method for total flavonoid content. Gallic acid was the only standard used in this study. The results obtained were compared to the positive control which is tsaang gubat that is already an established drug of the Bureau of Food and Drugs an approved by the Department of Health. 2. Experimental Materials The plants were collected and verified at the Bureau of Plant Industry. Leaves of tsaang gubat, banaba, malunggay, tubang-bakod, sambong and mangostan were used in the experiment. The reagents used were gallic acid, 2,2-diphenyl-1picrylhydrazil (DPPH) solution and Folin-Ciocalteu reagent. Staphylococcus aureus and Escherichia coli were used for antimicrobial activity of the plant samples done at the Natural Sciences Research Institute, University of the Philippines in Diliman. Preparation of gallic acid standards Two hundred fifty milligrams of dry gallic acid were dissolved in 1 ml of 95% ethanol and diluted to 500 ml with distilled water to prepare 0.5 mg/ml stock standard solution of gallic acid. The solution was stored in the freezer. Working standards were prepared between 0.0 mg/ml and 1 mg/l by diluting the stock solution with distilled water. Sample preparation Dried leaves were minced into the smallest pieces possible before subjected to liquid extraction. Ten grams of the minced samples were mixed with 100 ml methanol for 2 hours in an orbital shaker. The heterogeneous solution was centrifuged for 15 minutes. The extraction method was done in triplicate. The supernatant collected was filtered and dried. The amount of residue was determined in terms of weight (in mg). The residue was dissolved in 100 ml methanol and analyzed for its total polyphenol content, total flavonoid content and radical scavenging activity. The solution was filtered before analysis.

DPPH radical scavenging method A methanolic solution (0.1 ml) of sample extract was added at various concentrations to 3.9 ml (0.025 g L-1) of DPPH solution. The decrease in absorbance at 515 nm was determined continuously every 1 minute with a Perkin-Elmer UV-Vis Spectrophotometer until the reaction reached the plateau. The percentage of DPPH radical scavenging was calculated using the equation: Initial absorbance − Final absorbance % DPPH RSA = x 100 Initial absorbance Total polyphenol content The TPP content was determined by the Folin-Ciocalteu method. Plant extracts (0.5 ml) or gallic acid standard solutions were mixed with 2.5 ml of Folin-Ciocalteu reagent (FCR 1:10 dilution) and were left standing for 8 minutes at room temperature to allow for the Folin-Ciocalteu reagent to react completely with the oxidizable substances or phenolates. Two milliliters of Na2CO3 (7.5% solution in water) were added to destroy the residual agent. The absorbance was measured at 760 nm using a Lambda 40 UV-Vis Spectrophotometer (Perkin Elmer, USA) after incubating at room temperature for 2 hours. Results were expressed as milligrams of gallic acid equivalents (GAE) per 100 g fresh weights. Total flavonoid content Total flavonoid content was measured by aluminum chloride colorimetric assay. An aliquot (1ml) of stock solution or standard solution of gallic acid in methanol (0 mg/ml to 100 mg/ml) was combined with 4 ml double-distilled water. The amount of 0.3 ml 5% NaNO2 was added to the solution. After 5 minutes, 0.3 ml 10% AlCl3 was added and at the 6th minute, 2 ml of 1 M NaOH was added and the volume adjusted to 10 ml with double-distilled water. The solution was mixed well and the absorbance was measured against a reagent blank at 510 nm. Antimicrobial activity measurement Staphylococcus aureus and Escherichia coli were used for antimicrobial measurement of the plant samples at the University of the Philippines - Natural Sciences Research Institute. Microbial suspensions were prepared from 24-hour old cultures of the bacteria. The suspending medium used was 0.1% peptone water. One-tenth (0.1) ml aliquots of the bacteria were transferred into pre-poured nutrient agar. Five ml of the corresponding medium, melted and cooled to 45°C, was poured onto the agar plate and swirled to distribute the inoculum evenly on the agar surface. Three equidistant wells were made on the agar plate using a cork borer (10 mm). Two hundred µl of the sample were placed in each hole. The plates were incubated at room temperature. Nutrient agar plates were observed after 2448 hours. The clearing zones were calculated. The antimicrobial index (AI) was computed using the formula: AI =

Diameter

of clearing zone − diameter Diameter of clearing zone

of well

Statistical analysis Data were analyzed using one-way analysis of variance (ANOVA) test to calculate the significance of results in Microsoft Excel 2007. A difference was considered to be statistically significant when the P-value is less than 0.005 (*P < 0.005).

3. Results and discussion Extraction yield of the medicinal plant samples The different medicinal plant varieties used in the study contained levels of methanolic extracts ranging from 9.429 % of banaba to 17.316 % of malunggay as shown in Table 1. Sultana et al. (2009), conducted a study on the effects of four extracting solvents [absolute ethanol, absolute methanol, aqueous ethanol (ethanol: water, 80:20 v/v) and aqueous methanol (methanol: water, 80:20 v/v)] and two extraction techniques (shaking and reflux) on the antioxidant activity of selected medicinal plants including malunggay and achieved 9.61% to 17.9% yielded on different extracting solvents. Solvent extraction is the most frequently used technique for the isolation of plant antioxidant compounds. However, the extract yields and resulting antioxidant activities of the plant materials are strongly dependent on the nature of extracting solvent, due to the presence of different antioxidant compounds of varied chemical characteristics and polarities that may or may not be soluble in a particular solvent. Polar solvents are frequently employed for the recovery of polyphenols from a plant matrix. The most suitable of these solvents are (hot or cold) aqueous mixtures containing ethanol, methanol, acetone, and ethyl acetate.

There are abundant polyphenols in each plant species but relative amounts of flavonoids as shown in Figure 3.1, indicate that different classes of polyphenols, not just flavonoids, might be present in the leaves of the selected plants. Investigations by Igbinosa, et al., (2009) on the phytochemical screening of tubang-bakod stem bark extracts revealed the presence of saponins, steroids, tannins, glycosides, alkaloids and flavonoids. These compounds are known to be biologically active and therefore aid the antimicrobial activities of tubang-bakod. These secondary metabolites exert antimicrobial activity through different mechanisms. Herbs that have tannins as their main components are astringent in nature and are used for treating intestinal disorders such as diarrhea and dysentery. These observations therefore support the use of tubang-bakod in herbal cure remedies.

Table 1 Extraction Yield of the Different Medicinal Plants

Plant extracts Tsaang gubat Mangostan Sambong Tubang-bakod Banaba Malunggay

Extraction yield % 17.038 15.284 13.738 11.711 9.429 17.316

Total polyphenol content and total flavonoid content The total phenolic content and total flavonoid content of the medicinal plants were expressed as mg GAE/100grams and are listed in Table 3.2. The phenolic content for the methanolic extracts of the medicinal plants ranged from 507.40 to 980.93 mg GAE/100 g fresh weights. Tsaang gubat which is the positive control had the highest polyphenol content followed by mangostan, malunggay, banaba and tubang-bakod. The lowest polyphenol content was achieved by sambong. Flavonoids constitute the largest group of plant phenolics, accounting for over half of the eight thousand naturally occurring phenolic compounds (Manach et al., 2009). In this study, the total flavonoid contents of the medicinal plants ranged from 13.80 to 475.81 mg GAE/100 g. Tsaang gubat had the highest flavonoid content and tubang-bakod had the lowest. Statistical analysis showed that the medicinal plants are significantly different (*P<0.005), which affects the total phenolic and total flavonoid content. Table 2 Total Polyphenol and Total Flavonoid Content of the Medicinal Plant Extracts Plant extracts Tsaang gubat Mangostan Sambong Tubang-bakod Banaba Malunggay

TPP mg GAE/100g 980.93±5.9 965.73±8.6 507.40±9.7 546.4±6.70 865.00±5.6 885.07±5.9

TFC mg GAE/100g 475.81±8.3 462.63±2.4 111.37±9.7 13.80±2.6 26.30±8.3 279.50±5.8

Figure 1 Comparison of the TPP and TFC of the medicinal plants

A phytochemical investigation was done by Fazitalun et al., (2004) on the leaves of sambong by their chemical structures elucidated by means of elemental analyses and different spectroscopic methods, such as UV, IR, NMR and MS, which resulted in the isolation of 11 flavonoids (quercetin, rhamnetin, luteolin, luteolin-7-methylether, L-ascorbic acid, blumeatin, butylated hydroxyanisole, 5,7,3',5'-tetrahydroxyflavanone, tamarixetin, butylated hydroxytoluene, α-tocopherol, dihydroquercetin-4'-methylether and dihydroquercetin-7,4'dimethylether). The result indicates that flavonoid contents of different solvent extracts of sambong leaves are responsible for their antioxidant properties. Phenolics are often extracted in higher amounts in more polar solvents such as aqueous methanol/ethanol as compared with absolute methanol/ethanol (Sultana, et al., 2009). Previous reports suggest that phenolic content increases with the increase in leaf-age and is lower in early stages of leaf growth, gradually increasing with the maturity of leaves. It has also been reported that thermal processing conditions might result in the loss of natural antioxidants because heat may accelerate their oxidation and other degenerative reactions. Thus, heating temperature is of much consideration during processing. DPPH radical scavenging activity DPPH is a stable free radical with characteristic absorption at 515 nm and antioxidants react with DPPH and convert it to 2,2-diphenyl-1-picrylhydrazine. The DPPH radical has been widely used to test the free radical-scavenging ability of various natural products and has been accepted as a model

compound for scavenging free radicals in lipids. In the DPPH radical-scavenging method, a compound with high antioxidant potential effectively traps the radical, thereby preventing its propagation and the resultant chain reaction. The degree of discoloration from violet to yellow indicates the scavenging potential of the antioxidant extract, which is due to the hydrogen donating or radical scavenging ability (Verma, et al., 2009). High % DPPH radical scavenging activity would imply a high antioxidant activity. The DPPH radical scavenging of the methanolic extracts on Table 3.3 showed that tsaang gubat had the highest activity, which also had the highest polyphenol and flavonoid content followed by mangostan which also exhibited a high TPP and TFC, but the correlation of the results should be studied further. The high polyphenolic contents of the methanolic extracts could be responsible for the high antioxidant activity. Leong and Shui (2002) compared the total antioxidant capacity of twenty-seven fruits available in the Singapore market, including mangostan, using the ABTS and DPPH assays. Results showed that the mangostan extract had the eighth place in antioxidant efficiency. The DPPH radical scavenging activity of the six medicinal plants studied were significantly (*P < 0.005) different from each other. Table 3 DPPH Radical Scavenging Activity of the Medicinal Plant Extracts Plant extracts

% DPPH- RSA

Tsaang gubat Mangostan Sambong

69.36 54.88 40.32

±2.7 ±2.1 ±1.6

Tubang-bakod Banaba Malunggay

41.36 42.13 50.71

±3.1 ±2.9 ±3.1

It has been well established that free radical scavenging activity of plant extracts is mainly due to phenolic compounds. The weaker radical-quenching abilities observed for the compounds in this test may be ascribed to their molecular structures. Compounds having higher numbers of phenolic hydroxyl groups exhibited a stronger free radical-scavenging activity, which is in keeping with the notion that phenolic hydroxyl groups are able to donate hydrogens and those phenoxyl radicals, once formed, are stablilized by delocalization of electron. Sultana, that agroclimatic locations and seasons have profound effects on the antioxidant et al., (2009) concluded activity of malunggay leaves. Antioxidant activity of samples from cold areas was relatively higher than those from temperate regions. Antioxidant potential of malunggay leaves from Pakistan was quite comparable or higher than literature values for malunggay from other countries and some other potent antioxidants. This work shows that season and agroclimatic locations have profound effect on the antioxidant activity of malunggay leaves (Singh, et al., 2009). Antimicrobial activity Escherichia coli and Staphylococcus aureus enteropathogens are the common causes of diarrhea and were used as test organisms in this study. Thirty micrograms (30 ug) of chloramphenicol wereused as positive control having 6 mm in diameter for S. aureus and 8 mm for E. coli. Mangostan, tubang-bakod, sambong and tsaang gubat showed antimicrobial

activity against E. coli. Among all the extracts, tsaang gubat showed the highest antimicrobial activity against E. coli having a diameter clearing zone of 25 mm and antimicrobial index of 1.5. Malunggay did not show antimicrobial activity against E.coli and banaba showed only some thinning of E. coli. Mangostan, malunggay and sambong had slightly inhibitory effects against S. aures, but banaba, tubang-bakod and tsaang-gubat did not inhibit the growth of S. aureus. Results in Table 4 showed that both sambong and mangostan showed antimicrobial activity against Escherichia coli and Staphylococcus aureus. Medicinal plant extracts were significantly different (*P<0.005) on the antimicrobial activity with respect to E. coli and S. aureus. Table 4 Antimicrobial Activity of the Methanolic Extracts of the Medicinal Plants Escherichia coli Staphylococcus aureus UPCC1195 UPCC1143 Diameter Diameter of of clearing Antibacterial clearing Antibacterial zone Index zone Index Tsaang gubat 25.0 1.5±0.00 0.0 0±0.00 Mangostan 20.0 1±0.00 16.0 0.6±0.17 Tubang-bakod 20.0 1±0.00 0.0 0±0.00 Malunggay 0.0 0±0.00 11.3 0.1±0.06 Sambong 13.7 0.4±0.12 13.0 0.3±0.00 Banaba -(14) 0±0.00 0.0 0±0.00

Pedraza-Chaverri et al., (2008) isolated bioactive substances like xanthones from the fruits, pericarp and leaves extracts of mangostan which demonstrated that the extracts have antioxidant, antitumoral, antiallergic, anti-inflammatory, antibacterial, and antiviral activities. Villaseñor, et al., (2004) reported that the triterpene mixture, the major constituent isolated from tsaang gubat and which turned out to be anti-diarrheal, was inactive against Escherichia coli and possessed moderate activities against Staphylococcus aureus, Candida albicans, and Trichophyton mentagrophytes. The result is entirely different with the present study in which the extract of tsaang gubat is effective against Escherichia coli and has no inhibition with Staphylococcus aureus. In this study, tubang-bakod did not show activity against E.coli and showed inhibition against S. aureus. The findings of Lentz et al. (1998) were not in accordance with the results in which tubang-bakod did not have antimicrobial activity against E. coli and showed effectiveness on S. aureus. But in a study made by Adamu et al., (2005) tubang-bakod showed inhibition on both microorganisms. Some considerations must be established for the study of the antimicrobial activity of plant extracts, essential oils and the compounds isolated from them. This includes common parameters, such as plant material, techniques employed, growth medium and microorganisms tested. The solvent and the extraction system may both modify the final results. The most appropriate method would be that in which the extracts are the same as that used in folk medicine or phytotherapy, although in the lab the use of methanol or ethanol extract is much more common. The pH of compounds in dilutions also modifies the results, as can sometimes be observed when phenolic or carboxylic compounds are present in the extract. The composition of the growth medium can also influence the activity of the tested extracts or compounds (Rios & Recio, 2005).

Conclusion The selected traditional medicinal plants demonstrated abundant levels of polyphenols and it varies in their flavonoid contents. Tsaang gubat leaves achieved the highest total polyphenol, total flavonoid content and DPPH radical scavenging activity. Mangostan leaves could be an alternative for Tsaang gubat since it followed Tsaang gubat with the highest TPP, TFC and DPPH RSA and it showed inhibition on Escherichia coli and Staphylococcus aureus. The data gathered would certainly help to ascertain the potency of the six tested medicinal plant leaves as potential sources of natural antioxidants. Most of the methanolic extracts showed antimicrobial activity against Escherichia coli and had slightly inhibitory effects on Staphylococcus aureus. This has to be studied further and some general considerations must be considered such as the plant material, techniques employed pH, extraction methods, solvents, growth medium and microorganisms tested. The study of traditional medicinal plants as antimicrobial agents is necessary for gaining insight into the validity of their medicinal purposes. References Adamu, H., Abayeha, O., Aghoa, M., Abdullahi, A., Ubac, A., Dukkuc, H., Wufema, B. (2005). An ethnobotanical survey of Bauchi State herbal plants and their antimicrobial activity. Journal of Ethnopharmacology, 99, 1–4. Akao, Y., Nakagawa, Y., Iinuma, Munekazu., Nozawa, Y. (2008). Anti-Cancer Effects of Xanthones from Pericarps of Mangostan. Intrenational Journal Molecular Science, 9, 355-370 Alanis, A., Calzada F., Cervantes, J., Torres, J., Ceballos, G. (2005). Antibacterial properties of some plants used in Mexican traditional medicine for the treatment of gastrointestinal disorders. Journal of Ethnopharmacology, 100, 153–157. Balasundram, N., Sundram, K., Samman, S. (2006). Phenolic compounds in plants and agri-industrial by-products: Antioxidant activity, occurrence, and potential uses. Food Chemistry 99, 191–203. Basha, S., Francis, G., Makkar, H., Becker, K., Sujatha, M. (2009). A comparative study of biochemical traits and molecular markers for assessment of genetic relationships between Jatropha curcas L. germplasm from different countries. Plant Science 176, 812–823.

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